Method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli

A technology of green fluorescent protein and heterologous protein, which is applied in the field of extracellular secretion and expression of heterologous protein mediated by superfolded green fluorescent protein in Escherichia coli, which can solve the problems of complex operation, complicated operation process and low yield of target protein

Active Publication Date: 2017-04-26
HUBEI UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

Due to the limited size of the periplasmic space, disulfide bond mismatches sometimes occur, resulting in a small amount of correctly folded target protein, coupled with the barrier of the outer membrane of the cell, the output of the target protein secreted out of the cell is very small
[0003] According to literature reports, although by increasing the permeability of the outer membrane (ultrasound, adding Mg 2+ , Ca 2+ , EDTA, glycine and Triton X-100 and other chemical reagents, and lysozyme treatment, etc.), selection of gene-deficient strains (L-type bacteria), co-expression strategy (Kil protein), etc. can increase the amount of extracellular secretion of the target protein, but At the same time, it also introduces problems such as complicated operation, harsh secretion conditions, and slow production of E. coli, which is not suitable for large-scale production
[0004] Therefore, it is necessary to find new secreted proteins for the extracellular secretion and expression of heterologous proteins in Escherichia coli, so as to solve the shortcomings of the current secretory expression technology, such as complex operation process, signal peptide-mediated, low yield of target protein, etc. and suitable for large-scale production

Method used

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  • Method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli
  • Method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli
  • Method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli

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Embodiment 1

[0075] Superfolded green fluorescent protein (sfGFP) mediates the extracellular secretion and expression of toxic protein (antimicrobial peptide PG4) in Escherichia coli RosettaBlue. First, the constructed recombinant plasmid pET23a / sfGFP-PG4 was transformed into Escherichia coli competent cell Rosetta Blue strain, and cultured overnight at 37°C to obtain the recombinant strain. Next, pick a single colony and inoculate it in 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), culture it with shaking at 37°C, the OD value is between 0.5 and 0.6, add IPTG, the final concentration is 1mM, shake at 37°C Incubate for 8 hours. After the cultivation is completed, the bacterial cells and the medium supernatant are collected by centrifugation at 12000 RPM at 4°C. SDS-PAGE detection and analysis of fusion protein secretion and expression.

Embodiment 2

[0077]Superfolded green fluorescent protein (sfGFP)-mediated enzyme (β-N-acetylglucosaminidase H, Endo H) was secreted and expressed extracellularly in Escherichia coli Rosetta Blue. First, the constructed recombinant plasmid pET23a / sfGFP-Endo H was transformed into Escherichia coli competent cell Rosetta Blue strain, and cultured overnight at 37°C to obtain the recombinant strain. Next, pick a single colony and inoculate it in 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), culture it with shaking at 37°C, the OD value is between 0.5 and 0.6, add IPTG, the final concentration is 1mM, shake at 37°C Incubate for 8 hours. After the cultivation is completed, the bacterial cells and the medium supernatant are collected by centrifugation at 12000 RPM at 4°C. The samples were processed as follows: ①SDS-PAGE detection and analysis of the secretion and expression of the fusion protein; ②Referring to the detection method in the content of the invention, the e...

Embodiment 3

[0079] Superfolded green fluorescent protein (sfGFP) mediates the extracellular secretion expression of homotrimeric protease (human arginase-1, ARG1) in Escherichia coli Rosetta Blue. First, the constructed recombinant plasmid pET23a / sfGFP-ARG1 was transformed into Escherichia coli competent cell Rosetta Blue strain, and cultured overnight at 37°C to obtain the recombinant strain. Next, pick a single colony and inoculate it in 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), culture it with shaking at 37°C, the OD value is between 0.5 and 0.6, add IPTG, the final concentration is 1mM, shake at 37°C Incubate for 8 hours. After the cultivation is completed, the bacterial cells and the medium supernatant are collected by centrifugation at 12000 RPM at 4°C. The samples are processed as follows: ① SDS-PAGE detection and analysis of the secretion and expression of the fusion protein; ② referring to the detection method in the content of the invention, the ...

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Abstract

The invention provides a method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli. The method includes steps: 1) constructing a secretory expression carrier pET23a / sfGFP-GFP taking sfGFP (super-folded green fluorescent protein) as a secretory tag; 2) constructing a heterologous protein gene recombinant expression carrier; 3) respectively converting and fusing the expression carriers to escherichia coli competent cell Rosetta Blue to obtain a recombinant strain; 4) expressing, culturing and performing functional verification; 5) performing high-density fermentation of the recombinant strain. According to secretion characteristics of the super-folded green fluorescent protein, extracellular secretory expression of the heterologous protein in escherichia coli in a fusion protein form is realized without mediating through signal peptides, an operation process is simplified, high autocrine performance is realized, functional influences of tag protein on target protein are avoided, expression conditions can be quickly optimized, target protein yield is increased, and the method is suitable for large-scale production. In addition, an application field of sfGFP is expanded, and a novel method is provided for extracellular secretory expression of the heterologous protein in escherichia coli.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a superfolded green fluorescent protein (sfGFP)-mediated extracellular secretion expression method of a heterologous protein in Escherichia coli. Background technique [0002] The secretion and expression of heterologous proteins in Escherichia coli can be divided into periplasmic secretion and extracellular secretion. Periplasmic expression refers to the expression of recombinant protein transported from the cytoplasm to the periplasmic space mediated by signal peptides. Different types of signal peptides, such as PhoA, OmpA, OmpT, LamB, 6-lactamase, enterotoxin ST-II, LT-A, LT-B, Staphylococcus aureus protein A, human growth hormone signal peptide, etc., are often used. The expressed protein is transported from the cytoplasm to the periplasm. Since the periplasmic space is an oxidative gap, it can simulate the endoplasmic reticulum environment of eukaryotic cells. The n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12P21/02C12N9/24C12N9/78C12N9/88C12R1/19
CPCC07K14/00C07K2319/60C12N9/2402C12N9/78C12N9/88C12N15/70C12N2800/101C12Y302/01052C12Y305/03001C12Y401/01015
Inventor 张贞马立新卞璐易犁
Owner HUBEI UNIV
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